The extraction process of Japanese eel skin collagen was optimized using response surface methodology, and its physicochemical properties and allergenicity were investigated. Based on the single-factor experiment, the best extraction process was obtained based on the acid-enzymatic extraction method, with the material-liquid ratio, the concentration of extraction solution and the extraction time as the investigating factors and the collagen yield as the index. The results showed that the collagen contained five bands and belonged to type I collagen, and the percentages of essential amino acids and medicinal amino acids were 19.61% and 11.04%, respectively. The thermal transition temperature and thermal denaturation temperature of collagen were 38.5 and 28.3 ℃, respectively. The enzyme-linked immunosorbent reaction and simulated gastrointestinal fluid experiments showed that collagen has certain allergenic properties, providing a theoretical basis for further studies on eel skin collagen.